Abstract The overall goal of this proposal is to develop systems for efficient ex vivo maturation of functional human red blood cells (RBCs), which holds tremendous potential for addressing supply bottlenecks for delivering new therapeutic options to chronically transfused patients with severe alloimmunization and for novel strategies for delivering therapeutics via engineered RBCs. No approaches developed so far have shown the ability to produce both enucleated and mature RBCs that have reliable shelf-life in storage or viability upon transfusion. Our hypothesis is that in existing culture systems, late stage erythroblasts/early reticulocytes lose too much surface area during enucleation and membrane remodeling, leading to more spherical cells that are susceptible to hemolysis. We propose a biomimetic approach wherein cells are passed through microslit filters to recapitulate in vivo mechanical deformations that appropriately constrain cell area/volume, promote proper biconcave disc morphology and limit potentially harmful extensional stretching. This proposal brings together expertise in microfabrication at SiMPore Inc. with RBC biology and rheology at the University of Rochester. SiMPore will optimize micro-slit manufacturing methods and integrate microslit filter membranes into fluidic systems for RBC maturation testing at Rochester (Aim 1). Similar efforts will be carried out to test the capability of microslit filters to separate and concentrate mature/enucleated RBCs from nucleated precursors (Aim 2). Relevant experts will guide developing regulatory and commercialization plans so that the team is positioned to carry out clinical studies during Phase II. The developed RBC maturation tools can be married with existing large volume bioreactors for RBC precursor expansion so that final maturation using this approach can be scaled for practical volumes.